Thrombolytic agents and methods of treatment for thrombosis

ABSTRACT

The foregoing invention relates to a new microbubble preparation and thrombolytic therapy which relies on microbubbles and ultrasound for its lytic activity. The pharmaceutical composition of the invention comprises a liquid solution of microbubbles with an internal atmosphere enhanced with the perfluorocarbon gas which cavitate in the presence of an ultrasound field following intravenous injection or infusion of said composition into said host. For thrombolysis the area of a thrombus is exposed to an ultrasound field in the presence of the microbubbles and significant lysis is experienced. The method and pharmaceutical composition of the invention exhibit thrombolytic properties similar to those of other thrombolytic agents such as urokinase and are less toxic and are clot specific in that they do not introduce a systemic lytic state to a said animal.

FIELD OF THE INVENTION

This invention relates to a new and improved pharmaceutical compositionand method for treating thrombosis in animals. The methods andcomposition of the invention can be used as an anticoagulant therapy toinduce thrombolysis and to relieve trauma associated with obstruction ofsmaller vessels.

BACKGROUND OF THE INVENTION

Thrombosis, the formation and development of a blood clot or thrombuswithin the vascular system, while a life saving process when it occursduring a hemorrhage, can be life threatening when it occurs at any othertime. The thrombus can block a vessel and stop blood supply to an organor other body part. If detached, the thrombus can become an embolus andocclude a vessel distant from the original site.

In the healthy person there is a balance between clot formation(thrombosis) which is needed to minimize blood loss and to repair bloodvessels, and clot lysis (fibronolysis) which maintains the patency ofblood vessels. When thrombosis occurs without concomitant fibronolysiseffects can lead to strokes.

Traditional thrombolytic agents used are not clot specific and whilethey do break up the thrombus and facilitate fibronolysis they also putthe patient at significant risk as all clotting is inhibited and apatient could bleed to death from a small abrasion elsewhere. Currentthrombolytic agents include streptokinase which is derived fromBeta-hemolytic streptococci. When combined with plasminogen,streptokinase catalyzes the conversion of plasminogen to plasmin, theenzyme responsible for clot dissolution in the body. Three majorproblems encountered with the use of streptokinase therapy include itssystemic lytic effects coupled with a long half life. Because theanticoagulant activity of streptokinase is indiscriminent (non clotspecific) and prolonged (half life 10-18 minutes), bleeding is a commoncomplication which must be carefully monitored during 12 hours followingimmediately after administration. Further because streptokinase is abacterial protein, it is strongly antigenic and can produce a variety ofallergic reactions including anaphylaxis, particularly when administeredto a patient who has previously received streptokinase therapy or whohas had a recent streptococcal infection.

Another popular agent for use in treatment of thrombosis is urokinase,an enzyme protein secreted by the parenchyma cells of the human kidney.It acts to direct activation of plasminogen to form plasmin. This isdifferent from streptokinase which first forms a complex withplasminogen to activate plasmin to dissolve the clot. Urokinase is alsonon clot specific (activates circulating non clot bound plasminogen aswell as clot bound plasminogen) but has a shorter half life thanstreptokinase. Its administration is associated with fewer bleedingcomplications despite the fact that a systemic lytic state is alsoproduced. Urokinase is produced by the kidney and as such it is notantigenic and well suited for use if subsequent thrombolytic therapy isneeded. The major problem with urokinase is that it is difficult andexpensive to produce precluding its extensive clinical use.

The most recently developed drug in treating of thrombolysis isrecombinant tissue plasminogen activator. Approved by the FDA inNovember of 1987, tissue plasminogen activator (t-PA) is a naturallyoccurring enzyme (thus non antigenic) that is clot specific and has avery short half life (3-5 minutes). It converts plasminogen to plasminafter binding to the fibrin-containing clot. This clot specificityresults in an increased concentration and activity of plasmin at thesite of the clot, where it is needed. This characteristic of t-PAprevents the induction of the systemic lytic state that occurs withstreptokinase and urokinase activity. However the results of studiescomparing the streptokinase and t-PA show similar incidences of bleedingfollowing administration. Successful gene cloning has made sufficientquantities of t-PA available for clinical use, however, the recombinanttechnology necessary for its production have also resulted in aprohibitive cost. As can be seen a need in the art exists for athrombolysis therapy which is clot specific, which does not induce asystemic lytic state and which is inexpensive and non antigenic topatients.

SUMMARY OF THE INVENTION

According to the invention a thrombolytic therapy is provided which issite-specific and non-antigenic. The therapy involves the use of apharmaceutical composition which comprises microbubbles of a diameter ofabout 0.1 to 10 microns, the interior of which has been enhanced with aninsoluble gas such as fluorocarbon gas, helium or sulfur hexafluorideand which gas is encapsulated in a protein-coated shell. The inventionuses agents and methods traditionally used in ultrasound imaging and assuch provides a means for visualization of the clot as it is beinglysed. Quite unexpectedly it was found that the insoluble gasmicrospheres of the invention act themselves as a thrombolytic agent inthe presence of an ultrasound field and work as well as traditionalthrombolytic agents such as urokinase.

DETAILED DESCRIPTION OF THE INVENTION

Ultrasonic imaging has long been used as a diagnostic tool to aid intherapeutic procedures. It is based on the principle that waves of soundenergy can be focused upon an area of interest and reflected to producean image. Generally an ultrasonic transducer is placed on a body surfaceoverlying the area to be imaged and ultrasonic energy, produced bygenerating and receiving sound waves, is transmitted. The ultrasonicenergy is reflected back to the transducer where it is translated intoan ultrasonic image. The amount of characteristics of the reflectedenergy depend upon the acoustic properties of the tissues, and contrastagents which are echogenic are preferably used to create ultrasonicenergy in the area of interest and improve the imaging received. For adiscussion of contrast echographic instrumentation, see, DeJong and,"Acoustic Properties of Ultrasound Contrast Agents", CIP-GEGEVENSKONINKLIJKE BIBLIOTHEEK, DENHAG (1993), pp. 120 et seq.

Contrast echocardiography has been used to delineate intracardiacstructures, assess valvular competence, and demonstrate intracardiacshunts. Myocardial contrast echocardiography (MCE) has been used tomeasure coronary blood flow reserve in humans. MCE has been found to bea safe and useful technique for evaluating relative changes inmyocardial perfusion and delineating areas at risk.

Ultrasonic vibration has also been used in the medical field to increasethe absorption of various medicaments. For example in Japanese PatentKokai number 115591/1977 discloses that percutaneous absorption of amedicament is enhanced by applying an ultrasound vibration. U.S. Pat.Nos. 4,953,565 and 5,007,438 also disclose a technique of percutaneousabsorption of medicaments by the aid of ultrasonic vibration. U.S. Pat.No. 5,315,998 discloses a booster for drug therapy comprisingmicrobubbles in combination ultrasonic energy to allow the medicament todiffuse and penetrate at the site of interest.

Quite surprisingly applicant has demonstrated that a microbubblecomposition in combination with ultrasound therapy can act as athrombolytic medicament causing clot lysis at the site of a thrombus. Inthe presence of ultrasound the microbubbles themselves act as amedicament and are as effective as traditional thrombolytic agents suchas urokinase or t-PA. The pharmaceutical composition of the inventioncomprises a liquid containing microbubbles of an insoluble gas having adiameter of 0.1 to 10 microns. The microbubbles are formed by entrappingmicrospheres of a gas into a liquid. The microbubbles are made ofvarious gases preferably inert gases as xenon, krypton, argon, neon,helium, or fluorocarbon gases. The liquid includes any liquid which canform microbubbles. Generally any inert gas can be used. It must begaseous at body temperature and be nontoxic. The gas must also formstable microbubbles of average size of between about 0.1 and 10 micronsin diameter when the pharmaceutical composition is sonicated to formmicrobubbles. Generally perfluorocarbon gases such as perfluoromethane,perfluoroethane, perfluoropropane, perfluorobutane, perfluoropentane arepreferred. Of these gases, perfluoropropane and perfluorobutane areespecially preferred because of their demonstrated safety forintraocular injection in humans. They have been used in human studiesfor intraocular injections to stabilize retinal detachments (Wong andThompson, Opthamology 95:609-613). Treatment with intraocularperfluoropropane is considered to be the standard of care for treatmentof this disorder. The gases must also have a diffusion coefficient andblood solubility lower than nitrogen or oxygen which diffuse once in theinternal atmosphere of the blood vessel.

Other inert gases such as sulfur hexafluoride are also useful in theinvention provided they have a diffusion coefficient and bloodsolubility lower than nitrogen or oxygen. The agent of the invention isformulated in a pharmaceutically effective dosage form for peripheraladministration to the host in conjunction with ultrasound therapy.Generally such host is a human host, although other mammalian hosts suchas canine or equine can also be subject to this thrombolytic therapy.

In a preferred embodiment the pharmaceutical liquid composition of theinvention uses a liquid wherein the microbubbles are stabilized by afilmogenic denaturable protein coating. Suitable proteins includenaturally occurring proteins such as albumin, human gamma globulin,human apatransferin, Betalactose and urease. The invention preferablyemploys a naturally occurring protein but synthetic proteins may also beused. Particularly preferred is human serum albumin.

It is also preferred to use an aqueous solution containing a mixture ofa pharmaceutically accepted saccharide e.g., dextrose, in combinationwith the earlier described protein. In a most preferred embodiment thepharmaceutical liquid composition of the invention is the sonicatedmixture of commercially available albumin (human), U.S.P. solution(generally supplied as 5% or 25% by weight sterile aqueous solutions),and commercially available dextrose, U.S.P. for intravenousadministration. The mixture is sonicated under ambient conditions i.e.room air temperature and pressure and is perfused with an insoluble gas(99.9% by weight) during sonication.

In a most preferred embodiment the pharmaceutical liquid compositionincludes a two-fold to eight-fold dilution of 5% to 50% by weight ofdextrose and a 2% to 10% by weight of human serum albumin. Exemplary ofother saccharide solutions of the invention are aqueous monosaccharidesolution (e.g. having the formula 6CH6012 such as the hexos sugars,dextrose or fructose or mixtures thereof), aqueous disaccharide solution(e.g. having a formula C₁₂ H₂₂ O₁₁ such as sucrose, lactose or maltoseor mixtures thereof), or aqueous polysaccharide solution (e.g. solublestarches having the formula C₆ H₁₀ O₅ (n) wherein n is a whole numberinteger between 20 and about 200 such as amylase or dextran or mixturesthereof.

The microbubbles are formed by sonication, typically with a sonicatinghorn. Sonication by ultrasonic energy causes cavitation within thedextrose albumin solution at sites of particulate matter or gas in thefluid. These cavitation sites eventually resonate and produce smallmicrobubbles (about 7 microns in size) which are non-collapsing andstable. In general, sonication conditions which produce concentrationsof greater than about 4×10⁸ m of between about 5 and about 6 micronmicrobubbles are preferred. Generally the mixture will be sonicated forabout 80 seconds, while being perfused with an insoluble gas.

A second method of preparation includes hand agitating 15±2 ml ofsonicated dextrose albumin with 8±2 ml of perfluorocarbon gas prior toson±cation. Son±cation then proceeds for 80±5 seconds. Generally thepharmaceutical liquid composition is injected into the area of thethrombosis or close thereto and then ultrasound is applied.

These microbubble sizes are particularly ideal since a microbubble musthave a mean diameter of less than 10 microns and greater than 0.1 to besufficient for transpulminary passage, and must be stable enough toprevent significant diffusion of gases within the microbubble followingintravenous injection and during transit to the thrombosis site. Themethod preferred for practicing the anti thrombosis therapy of theinvention involves obtaining a pharmaceutical liquid agent of theinvention, introducing said agent into a host by intravenous injection,intravenously (i.v. infusion), percutaneously or intramuscularly.Injection is such that the area of the thrombus is perfused with thepharmaceutical composition. Next ultrasound is applied thereto using asuitable Doppler or ultrasound echo apparatus so that the field ofultrasound encompasses the thrombus. The ultrasound signal activates themicrobubbles so that the microbubbles themselves act as a thrombolyticagent.

The desired ultrasound is applied by conventional ultrasonic deviceswhich can supply an ultrasonic signal of 20 Khz to several Mhz and isgenerally applied from about 3 to about 5 Mhz.

In the most preferred embodiment the agent of the invention is aperfluorocarbon enhanced sonicated dextrose albumin solution comprisedof a sonicated three-fold dilution of 5% human serum albumin with 5%dextrose. During sonication, the solution is perfused withperfluorocarbon gas for about 80 seconds which lowers the solubility anddifusivity of the microbubble gas. The resulting microbubbles areconcentrated at room temperature for at least about 120±5 minuteswherein the excess solution settles in the sonicating syringe. Theexcess solution is expelled and the concentrated microbubbles aretransferred to a sterile syringe and injected parenterally into amammal, near the site of the thrombus.

Methods of ultrasonic imaging in which microbubbles formed by sonicatingan aqueous protein solution are injected into a mammal to alter theacoustic properties of a predetermined area which is then ultrasonicallyscanned to obtain an image for use in medical procedures is well known.For example see U.S. Pat. No. 4,572,203, U.S. Pat. No. 4,718,433 andU.S. Pat. No. 4,774,958, the contents of each of which are incorporatedherein by reference.

It is the use of these types of contrast agents as a pharmaceuticalcomposition and application of ultrasound as an anti thrombosis therapythat is the novel improvement of this invention. Blood clots whentreated with the microbubble composition and therapy of this inventionwere shown to decrease in size by a percentage equal to that oftraditional thrombolytic agents such as urokinase. According to theinvention, it was shown that treatment with decafluorobutane sonicateddextrose albumin microbubbles and subsequent application of ultrasoundresulted in a higher percentage of clot reduction than treatment withurokinase alone. The combination of perfluorocarbon enhanced sonicateddextrose albumin microbubbles (PESDA) and ultrasound resulted inincreased clot lysis from that of ultrasound alone or over use of PESDAalone.

This is particularly significant as the microbubble anti-thrombosistherapy can reduce any toxic effects of persons who cannot otherwise usetraditional thrombolytic agents such as urokinase. According to theinvention the thrombosis can be treated simply with ultrasound incombination with a microbubble pharmaceutical composition of theinvention and the protein substance such as human serum albumin iseasily metabolized within the body and excreted outside and hence is notharmful to the human body. Further gas trapped within the microbubblesis extremely small and is easily dissolved in blood fluid,perfluoropropane and perfluorobutane have long been known to be safe inhumans. Both have been used in humans for intra ocular injections tostabilize retinal detachments. Wong and Thompson, Opthalmology95:609-613. Thus the anti thrombosis agents of the invention areextremely safe and nontoxic for patients.

The following examples are for illustration purposes only and are notintended to limit this invention in any way. These examples demonstratethe effect of the pharmaceutical compositions and therapy of theinvention. In all the following examples, all parts and percentages areby weight unless otherwise, all dilutions are by volume.

EXAMPLES Preparation of Anti Thrombosis Pharmaceutical Agent

Albumin (human) USP, 5% solution (hereinafter referred to as "albumin")and dextrose USP, 5% solution (hereinafter referred to as "dextrose")were obtained from a commercial source. The sonicating system used forsonication was a Heat System Ultrasonic Processor Model XL2020 (HeatSystems Inc., Farmingdale, N.Y.). The 1/2 inch horn transducer was aresonating piezoelectric device. The 1/2 inch sonicating horn tip wassterilized prior to each sonication.

Sonication of Samples

Sixteen milliliter aliquots of albumin diluted 1:3 with dextrose weredrawn up into a 35 cc "Monoject" syringe (Becton Dickinson and Company,Rutherford, N.J.) and sonicated for 80±1 seconds. The "Leur-Lok" of the35 milliliter syringe was then attached to a stopcock. After mixing thedextrose albumin solution by hand for about 7 to about 10 seconds, theplunger was removed from the top of the syringe. The sterile sonicatinghorn was then lowered into the open end of the syringe until at thesurface of the albumin-dextrose solution. The solution was placed at thehorn tip and manually held at this position while continuouslysonicating at a frequency of 20,000 Hz and a power output of 210 W for80±1 seconds to form a stable microbubble solution.

Gas Perfusion of Samples

A second method of preparation includes hand agitating 15±2 ml ofsonicated dextrose albumin with 8±2 ml of perfluorocarbon gas prior tosonication. Sonication then proceeds for 80±5 seconds. Generally thepharmaceutical liquid composition is injected into the area of thethrombosis or close thereto and then ultrasound is applied.

The dextrose albumin mixture was exposed to either perfluoropropane orperfluorobutane gas (Commercial Grade, 99.9% by weight) by handagitating 15±2 ml of sonicated dextrose albumin with 8±2 ml ofperfluorocarbon gas prior to sonication. Theperfluorocarbon/dextrose-albumin mixture was then sonicated for 80±5seconds. The total volume of perfluorocarbon-enhanced sonicated dextrosealbumin produced with this formulation was 25±2 milliliters. Thesesamples were then used for intravenous injection.

Microbubble Analysis

Microbubble size and purity was determined using hemocytometry.Microscopic inspection of the microbubbles was performed to determine ifany coalescent microbubbles were present in the solution. Microbubbleconcentration was determined using a Coulter Counter. The antithrombosis pharmaceutical agent was rejected for use if any of thefollowing conditions are present: the mean microbubble size was 4.0 to6.0 microns; coalesced microbubbles or strands were detected by lightmicroscopy; or the mean microbubble concentration was less than 0.8×10⁹or greater than 1.5×10⁹ microbubble/milliliter. The sample was alsorejected if the number of microbubbles greater than 10 microns in thesample was greater than 4%.

All samples were stored in 35 milliliter syringes until time ofinjection. All solutions were given within 36 hours of production. Allsamples were prepared in a laminar flow hood.

Example 1

In Vitro Clot Lysis Using PESDA and Ultrasound

The pharmaceutical compositions and method of the invention were shownto reduce the size of blood clots according to the following in vitroprotocol. The protocol is known in the art and is predictive of successin vivo Sehgal, "Ultrasound-Assisted Thrombolysis", Invest-Radiol.,October 1993, Vol. 28, No. 10:939-43. 2 ml aliquots of freshly drawnwhole blood were placed into a 10 cc plunger inverted syringes. Theblood was then incubated for 2 hours at 37° C. After incubated, thesyringes were removed from the water bath and left at room temperatureuntil treatment. Upon treatment the serum was decanted from the clot bypouring the contents of the syringe over a wire mesh screen. The clotwas then dried by rolling in the screen and blotting. The clot was thenweighed and placed back into the syringe with lytic fluid (microbubblepharmaceutical composition of the invention). Samples without treatmentwere incubated at 37° C. in a water bath for 20 minutes. Samples withtreatment involved placement of the ultrasound horn approximately 2 mlin solution and ultrasound was applied for 2 minutes. After 2 minutesthe clot was incubated for 18 minutes at 37° C. Again the fluid wasdecanted, the clot was rolled and blotted on the bottom of the screen todry and the clot was weighed subsequent to therapy.

Several experiments were run using this protocol and the results areshown in the following tables.

    ______________________________________                                        Experiment #1                                                                 With Ultrasound   Without Ultrasound                                                         % clot lysis            % clot lysis                           Sample  n      Average    Sample  n    Average                                ______________________________________                                        saline  4      7.4        saline  4    8.7                                    urokinase                                                                             4      46.3       urokinase                                                                             5    17.9                                   PESDA   4      15.3       PESDA   4    3.1                                    ______________________________________                                    

    ______________________________________                                        Experiment #2                                                                 With Ultrasound   Without Ultrasound                                                         % clot lysis            % clot lysis                           Sample  n      Average    Sample  n    Average                                ______________________________________                                        saline  4      33.3       saline  3    4.1                                    urokinase                                                                             4      54.9       urokinase                                                                             4    12.4                                   PESDA   6      58.7       PESDA   4    3.1                                    ______________________________________                                    

    ______________________________________                                        Experiment #3                                                                 With Ultrasound   Without Ultrasound                                                         % clot lysis            % clot lysis                           Sample  n      Average    Sample  n    Average                                ______________________________________                                        saline  4      10.9       saline  4    7.8                                    urokinase                                                                             4      45.1       urokinase                                                                             4    17.7                                   PESDA   4      50.9       PESDA   4    4.1                                    ______________________________________                                    

    ______________________________________                                        Experiments 1, 2 and 3 combined                                               With Ultrasound   Without Ultrasound                                                         % clot lysis            % clot lysis                           Sample  n      Average    Sample  n    Average                                ______________________________________                                        saline  12     12.9       saline  11   6.9                                    urokinase                                                                             12     48.8       urokinase                                                                             13   16                                     PESDA   14     44.0       PESDA   12   3.4                                    ______________________________________                                    

As can be seen from the foregoing tables, when all data is combined withover 10 separate experiments, ultrasound in combination withperfluorobutane enhanced, sonicated dextrose albumin microspheresdemonstrated an average percent clot lysis that was approximately equalto that which resulted from urokinase in combination with ultrasound.

As can be seen quite unexpectedly, in the presence of ultrasound, PESDAmicrobubbles work as a thrombolytic agent to reduce the size of athrombous at a level which rivals that of traditional thrombolyticagents such as urokinase.

Example 2 (Prophetic)

For humans the anti thrombosis therapy includes doses of the liquidpharmaceutical composition, PESDA, from about as small as 0.0025 up to0.1 ml/kg given depending on the ultrasonic procedure used. The contrastagent is given by peripheral intravenous infusion over about 1-25minutes (the dose range is patient specific. Large patients may requireslightly higher doses to produce equivalent thrombolysis). Generally inone protocol a patient will receive a 0.01 ml/kg of perfluorocarbonenhanced sonicated dextrose albumin or 0.0015 ml/kg perfluorobutanesonicated dextrose albumin as the initial injection. If this fails toproduce significant clot lysis, the dose could then be doubled. Dosingprotocols would be similar to those used for ultrasound imaging and aredisclosed in Wyman, Arthur E. "Principles and Practice ofEchocardiography", Lee & Febiger, Malvern, Pa. (1994 2nd Edition). Anyultrasound device can be used including the commercially availableHewlett Packard Sonus 1500 Phased Ray Imaging System (Hewlett Packard,Andover, Mass.). The patient is exposed to ultrasound for a timesufficient to experience significant clot lysis and generally will befrom about 1 to about 25 minutes. Thrombolysis can be monitored byviewing with conventional angiography, using radiographic dyes, or otheraccepted methods.

What is claimed is:
 1. A method of treating thrombosis in animalscomprising the steps of:introducing a solution consisting essentially ofa mixture of dextrose and albumin to said animal by intravenousinjection near a thrombus site and thereafter applying ultrasound tosaid site, said dextrose and albumin mixture comprising a plurality ofmicrobubbles with a diameter of from about 0.1 to 10 microns, saidmicrobubbles having a protein coated shell and an internal atmospherecomprising a gas which is insoluble in blood.
 2. The method of claim 1wherein said dextrose and albumin mixture is a solution of dextrose. 3.The method of claim 1 wherein said protein coated shell is an albumincoated shell.
 4. The method of claim 1 wherein said insoluble gas isselected from the group consisting of perfluoromethane, perfluoroethane,perfluoropropane, perfluorobutane and perfluoropentane.
 5. Thepharmaceutical composition of claim 4 wherein said perfluorocarbon gasis perfluorobutane.
 6. The pharmaceutical composition is claim 4 whereinsaid perfluorocarbon gas is perfluoropropane.
 7. The method of claim 1wherein said pharmaceutical compositions are created by the followingsteps:mixing an aqueous solution comprising 2% to about 10% by weight ofhuman serum albumin diluted about two fold to about eight fold with 5%to 50% by weight dextrose; and exposing said solution to a sonicationhorn to create cavitation at particulate sites in said solutiongenerating stable microspheres from about 0.1 to 10 microns in diameter.8. The method of claim 7 wherein said dilution of albumin with dextroseis a 3-fold dilution.
 9. The pharmaceutical composition of claim 7wherein said human serum albumin is a 5% by weight solution.
 10. Themethod of claim 7 wherein said dextrose is a 5% by weight solution. 11.A method for treating thrombosis in animals comprising:(a) obtaining apharmaceutical composition which consists essentially of:(i) an aqueousalbumin-dextrose solution containing between about a two fold and aboutan eight fold dilution of between about 5% to 50% by weight dextrose andbetween about 2% to about 10% by weight human serum albumin; and (ii)microbubbles, having a protein coated shell and a gaseous content ofwhich contain an amount of perfluorocarbon gas effective for achieving astable concentration of about 5×10⁸ microspheres per ml; (b) introducingsaid pharmaceutical composition to said thrombus; and (c) exposing saidmicrobubbles and said thrombus to an ultrasound field for a timesufficient to lyse said thrombus.
 12. The method of claim 11 whereinsaid step of introducing said composition to said thrombus is byintravenous injection.
 13. The method of claim 11 wherein said dextroseis a 5% solution.
 14. The method of claim 11 wherein said protein coatedshell is an albumin coated shell.
 15. The method of claim 11 whereinsaid perfluorocarbon gas is selected from the group consisting ofperfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane andperfluoropentane.
 16. The pharmaceutical agent of claim 15 wherein saidperfluorocarbon gas is perfluorobutane.
 17. The pharmaceutical agent isclaim 15 wherein said perfluorocarbon gas is perfluoropropane.
 18. Themethod of claim 11 wherein said pharmaceutical compositions are createdby the following steps:mixing an aqueous solution comprising 2% to about10% by weight of human serum albumin diluted about two fold to abouteight fold with 5% to 50% by weight dextrose; and exposing said solutionto a sonication horn to create cavitation at particulate sites in saidsolution generating stable microspheres from about 0.1 to 10 microns indiameter.
 19. The method of claim 18 wherein said dilution of albuminwith dextrose is a 3-fold dilution.
 20. The pharmaceutical agent ofclaim 16 wherein said human serum albumin is a 5% by weight solution.21. The method of claim 18 wherein said dextrose is a 5% by weightsolution.
 22. A method of treating thrombosis in animals comprising thesteps of:introducing a solution of protein encapsulated, blood insolublegas filled microbubbles to said animal by intravenous injection near athrombus site, said solution comprising dextrose, albumin and amedicament wherein said medicament is other than a thrombolytic agent,and thereafter applying ultrasound to said site.